CN109983130B

CN109983130B - Methods of making electrotransport competent yeast cells and methods of using these cells

Info

Publication number: CN109983130B
Application number: CN201780069926.8A
Authority: CN
Inventors: 塞巴斯蒂安·邦克; 多米尼克·莫勒; 菲力士·尤佛多本
Original assignee: Immatics Biotechnologies GmbH
Current assignee: Immatics Biotechnologies GmbH
Priority date: 2016-11-15
Filing date: 2017-11-13
Publication date: 2023-10-20
Anticipated expiration: 2037-11-13
Also published as: TW201819621A; JP2019531751A; MA46835A; AR110087A1; ES2952039T3; PH12019501001A1; AU2017360666C1; MY191776A; CN109983130A; US10683495B2; TWI720268B; EP3541947B1; US20210355478A1; US20200263162A1; JP7133226B2; EP3541947A1; US20180135039A1; BR112019008385A2; AU2017360666B2; US11104894B2

Abstract

The present invention relates to improved yeast transformation of yeast cells, and to a library of yeast cells derived from such transformation. More specifically, the invention relates to yeast transformation by electroporation.

Description

Methods of making electrotransport competent yeast cells and methods of using these cells

Technical Field

The present invention relates to improved yeast transformation of yeast cells, and to a library of yeast cells derived from the transformation thereof. More specifically, the invention relates to yeast transformation by electroporation.

Background

For many years, the cornerstone for cancer treatment has been surgery, chemotherapy and radiation therapy. Targeted therapies have also become the standard treatment for many cancers in the last decade, such as imatinibAnd trastuzumab +>Is a drug that targets cancer cells by targeting specific molecular changes that are primarily seen in these cells.

Today, it is exciting to use more and more immunotherapy treatment, i.e. to use the strength of the patient's immune system to combat the disease. One approach to immunotherapy involves the genetic engineering of patient's autoimmune cells to recognize and attack their tumors. This method, known as Adoptive Cell Transfer (ACT), has produced some significant therapeutic effects in patients with advanced cancer.

While natural T Cell Receptors (TCRs) are generally expected to have sufficiently high affinity to achieve therapeutic efficacy, so-called affinity maturation is generally expected/required to elicit an effective immune response in vivo when developing therapeutic TCRs or derivatives thereof, such as soluble TCRs (stcrs).

To achieve the mature state, a method of yeast surface display technology is generally applied. However, in order to generate libraries with sufficient diversity, efficient yeast transformation methods are required.

It has long been desirable to identify TCRs that consist essentially of native alpha and beta chain sequences and that specifically bind to a particular antigen, so that, for example, TCRs or soluble analogues thereof can be developed, providing a basis for potential therapeutic agents. Antigens recognized by the above identified TCRs may be associated with diseases such as cancer, viral infections, autoimmune diseases, parasitic infections and bacterial infections. Thus, these therapies may be used to treat the disease.

Furthermore, once natural or native TCRs are found and their sequences are determined, mutations may be introduced as required which lead to an increase in affinity or half-life, as described in WO 2012/01301301313. Traditionally, attempts to find TCRs that specifically bind disease-associated antigens (e.g., cancer viral, autoimmune, or bacterial antigens) have been limited by the use of blood samples drawn from volunteer donors. These samples were used to isolate T cells and their corresponding TCRs that bind to disease-associated antigens. This method generally requires at least 20 donors. This process is lengthy and labor intensive and does not guarantee the discovery of antigen-binding T cell receptors. When functional T cell receptors are recognized, they typically have a weak affinity for the antigen, low specificity, and/or improperly fold in vitro. The diversity of T cells that can be screened is limited by the diversity of T cells in the donor. Some disease-associated antigens, including most cancer antigens, are self-antigens; since TCRs recognizing self-antigens are removed by thymus selection, TCRs specific for disease-related antigens may not be present in the natural composition of the donor or may have weak affinity for the antigen.

Attempts to design new TCR libraries with antigen binding specificity have been ongoing for several years. TCR libraries are far more difficult to create than homogeneous antibody libraries because TCR chains are less stable and often not correctly displayed. Construction of TCR libraries is very complex. Preferably, CDR3 length variants (found in natural lineages) are retained. A significant portion of either library is depleted of stop codons, frame shift, folding problems, and TCR chain combinations that may not bind to the HLA complex at all. Given the large number of variable alpha and beta genes and the J and D genes, the chance of producing and identifying functionally folded alpha and beta chains that together form a TCR that binds to an antigenic peptide and has the desired specificity is extremely low.

The utility of methods for generating nucleic acid libraries and recombinant products (e.g., pharmaceutical proteins) produced thereby in eukaryotic systems (e.g., yeast) provides significant advantages over the use of prokaryotic systems such as (e.g., e.coli). Yeast can generally grow to a higher cell density than bacteria and is readily adaptable to continuous fermentation processes. However, the development of yeast species as host/vector systems for the production of recombinant products and libraries is severely hampered by the lack of knowledge regarding transformation conditions and the appropriate methods for stable introduction of foreign nucleic acids into yeast host cells.

Among the various electrical and biological parameters that promote cell electrotransformation, DNA adsorption to the cell surface is one of them. The low intensity of the alternating electric field may also promote DNA transfer into E.coli, likely by electrical stimulation of DNA permeases. Evidence has been accumulated for the major electrodiffusion or electrophoretic effects of electroporation gene transfer of polyelectrolyte DNA. Electroosmosis and membrane invagination facilitated by electroporation have also been reported.

Application of an electric field across the yeast cell membrane can create transient pores critical to the electroporation process. The electroporator signal generator provides a voltage (in kV) across the gap (cm) between the electrodes. The potential difference defines the so-called electric field strength, where E is equal to kV/cm. Each cell has its own critical field strength for optimal electroporation. This is determined by the cell size, membrane composition and individual characteristics of the cell wall itself. For example, mammalian cells typically require an electric field strength of 0.5-5.0kV/cm for cell death and/or electroporation to occur. Generally, the field strength required is inversely proportional to the cell size.

EP2257638A1 relates to a method for transforming yeasts by electroporation. These methods include a combination of lithium acetate (LiAc) and Dithiothreitol (DTT) as cell regulators, both of which are used to increase the frequency of yeast transformation. As shown in table 2, the efficiency was reduced by 93.3% and 85.7% after the DTT or LiAc pretreatment was removed, respectively.

Similarly, smith et al (in T Cell Receptor Engineering and Analysis Using the Yeast Display platform. Methods Mol biol.2015; 1319:95-141), disclose studies on TCR binding to antigen as a peptide-MHC (pepMC) ligand. There has been interest in engineering TCR affinities to use such molecules in a similar manner to antibodies. To engineer TCRs and more rapidly analyze their binding characteristics, a yeast display system was used as a platform. Expression and design of a single chain form TCR similar to an antibody scFv fragment allows TCR affinity maturation engineering against a variety of possible pephc ligands. In addition, yeast display platforms allow one to rapidly generate TCR variants with multiple binding affinities and analyze the specificity and affinity without the need to purify soluble forms of TCR. Methods for single chain TCR design and analysis using yeast display technology are presented.

Yeast libraries do not reach the size or efficiency that phage libraries have achieved, typically a maximum phage library size of 10 ¹⁰ To 10 ¹¹ While a typical yeast library size is 10 ⁷ . Although recent advances in electroporation Protocols (see Chao, nature Protocols 1 (2): 755-768 (2006)) can achieve a maximum of 5x 10 in a single transformation ⁷ A yeast library of size. The yeast library sizes achieved to date are still significantly lower than the 10 conventionally achievable with phage display libraries ¹⁰ To 10 ¹¹ The size, this expression is still correct.

The method and disclosure described above are used to convert 10 while achieving higher and higher conversion efficiencies ⁶ To 10 ⁷ Multiple small libraries of a range of sizes accumulate to form 10 ⁸ To 10 ⁹ Large combinatorial libraries of a range of sizes are still laborious, time consuming and labor intensive.

Yeast display library selection using magnetic beads and fluorescence-initiated cell sorting methods provides an efficient and sensitive method to enrich for specific binders targeting antigens, especially their compatibility with fluorescence-initiated cell sorting (FACS). However, the limited size of typical yeast display libraries hampers the advantages of this selection due to the low transformation efficiency of yeast cells.

Therefore, a method for efficiently producing a protein library (for example, a TCR library) using yeast is required.

Disclosure of Invention

In one aspect, the invention provides a method of preparing an electrotransport competent yeast cell comprising the steps of: a) Growing yeast cells to OD ₆₀₀ A value of about 1.0 to 2; b) Washing the cells with cold water; c) By a process comprising sorbitol and CaCl ₂ Is used for washing cells; d) Incubating the cells in a solution comprising lithium acetate and tris (2-carboxyethyl) phosphine (TCEP); e) By a process comprising sorbitol and CaCl ₂ Is used for washing cells; f) Resuspending the cells in a solution comprising sorbitol; and g) optionally, storing the cells appropriately.

Accordingly, the present invention provides a method for efficiently transforming yeast cells, for example, for generating an improved library of yeast cells. The method of the present invention eliminates a significant bottleneck in the application of yeast display technology as a practical tool, allowing access to larger TCR diversity spaces that have not been explored before.

Another aspect of the invention relates to a method of transfecting an inductively receptive yeast cell comprising the steps of: a) Providing an electrotransport competent yeast cell according to the method of the invention; b) Washing the cells with a cold solution comprising sorbitol; c) Mixing the cells with DNA to be transfected to form an electroporation premix; d) Transferring the electroporation pre-mix to a suitable electroporation cuvette; and e) electroporating the cells between about 2.5kV/cm and about 12.5kV/cm for about 2 to about 5ms.

In the method of the invention, it is preferred that the DNA is linear or circular. More preferably, in the method of the invention, the DNA comprises a library of DNA fragments encoding a library of related proteins, for example a library of DNA in the form of a yeast surface display library. Most preferably, in the methods of the invention, the display library is a T Cell Receptor (TCR) library.

Surprisingly, the Applicant has found that by using tris (2-carboxyethyl) phosphine (TCEP) as reducing agent, the conversion efficiency of the process of the invention is higher than when DTT is used, for example higher than 1X 10 ⁸ Each yeast transformant/μg vector DNA, preferably higher than 2X 10 ⁸ Each yeast transformant/. Mu.g vector DNA.

Another aspect of the invention relates to a method for producing an improved library of target protein yeasts, for example a library in the form of a yeast surface display library, comprising the steps of: a) Providing a transfected yeast cell according to the method of the invention; b) Transfected cells were diluted in sorbitol solution: in a 1:1 mixture of growth media; c) Resuspending the cells in a suitable growth medium; d) Optionally, performing dilution to calculate diversity, and plating the dilution on a kanamycin-containing SD-CAA plate; and e) transferring said library to a suitable growth medium and expanding said library obtained by each electroporation; and f) optionally, storing the extended library appropriately.

Preferably, the method according to the invention, wherein the display library is a T cell receptor library. Preferably, the method according to the invention, wherein the library has a diversity of greater than about 10 ¹² 。

Detailed Description

In the context of the present invention, the term "expression vector" refers to a DNA construct comprising an Autonomous Replication Site (ARS), a transcription initiation site, and at least one structural gene encoding a protein to be expressed in a host organism. The replication site or origin is any DNA sequence that controls cloning and replication of the expression vector. Expression vectors will also typically contain suitable control regions, such as one or more enhancers and/or promoters, repressors and/or silencers, and terminators that control the expression of the protein in the host yeast. The expression vector according to the invention may also contain a selectable marker comprising the essential genes described herein. The expression vector also optionally contains other selectable markers that are widely available and well known to those of skill in the art. Expression vectors are one type of vector. The vector may optionally include one or more ARS sequences (elements) from one or more yeast strains.

The term "operably linked" refers to DNA segments that are formulated such that they act in concert for their intended purposes, e.g., transcription starts at the promoter and proceeds through the coding segment until a terminator is reached.

The term "transformation" or "transfection" refers to the introduction of DNA or other nucleic acid into a recipient yeast host cell to alter the genotype.

The term "transformant" or "transformed cell" refers to a recipient yeast host cell that has undergone transformation and its progeny.

Unless otherwise indicated, "about" shall be +/-10% of the given value.

Vectors useful in the electroporation methods of the invention include the pYD vector, any other vector that can be propagated by yeast cells or nucleic acids in general, and constructs derived therefrom. The expression vectors of the invention may be based on any type of vector, as long as the vector can transform, transfect or transduce a host yeast cell. In a preferred embodiment, the expression vector is based on a yeast plasmid, in particular a plasmid from Saccharomyces cerevisiae. After transformation of yeast cells, the foreign DNA encoding the library sequences is taken up by the cells and subsequently expressed by the transformed cells.

More preferably, the expression vector may be a yeast bacterial shuttle vector that can be propagated in E.coli or yeast (Struhl, et al (1979) Proc.Natl. Acad.Sci.). Incorporation of E.coli plasmid DNA sequences (e.g., pBR 322) facilitates quantitative preparation of vector DNA in E.coli, thereby allowing efficient transformation of yeast.

The type of yeast plasmid vector that can be used as a shuttle may be a replicative vector or an integrative vector. A replication vector is a yeast vector that is capable of mediating its self-maintenance independent of yeast chromosomal DNA due to the functional origin of DNA replication. The integrating vector relies on recombination with chromosomal DNA to promote replication and thus to continue to maintain the recombinant DNA in the host cell. The replication vector may be a 2 micron based plasmid vector in which the DNA replication origin is derived from an endogenous 2 micron plasmid yeast. Alternatively, the replication vector may be an Autonomous Replication (ARS) vector, wherein the "distinct" origin of replication is derived from the chromosomal DNA of the yeast. Optionally, the replication vector may be a Centromere (CEN) plasmid carrying, in addition to one of the above-mentioned DNA origins of replication, a yeast chromosomal DNA sequence known to contain centromeres.

The vector may be transformed into yeast cells in a dead-loop or linear form. Transformation of yeast by integrating vectors, while genetically stable, may be inefficient when the vector is in a compact circular form (e.g., only about 1-10 transformants per μg of DNA are produced). Linearized vectors having free ends in a DNA sequence homologous to yeast chromosomal DNA transform yeast with greater efficiency (100-1000 fold), and the transformed DNA is typically found integrated into a sequence homologous to the cleavage site. Thus, by cleaving the vector DNA with the appropriate restriction endonuclease, the transformation efficiency can be increased and targeted to the chromosomal integration site. Integrative transformation can be suitable for the genetic modification of brewery yeasts, provided that the transformation efficiency is sufficiently high and the target DNA sequence for integration is in a region which does not disrupt the genes necessary for host cell metabolism.

Yeast strains that can be transformed by the electroporation method of the present invention include Saccharomyces cerevisiae (e.g., saccharomyces cerevisiae) and Schizosaccharomyces pombe (e.g., schizosaccharomyces pombe). In one embodiment, the yeast cell is a diploid yeast cell. Alternatively, the yeast cells are haploid cells, e.g., the "a" and "a" strains of yeast haploid cells.

The "T cell receptor library" in the context of the present invention may comprise suitable portions of the human and/or mutated human TCRs to be screened, preferably in single chain form, such as for example: vβ -linker-vα single chain (scTCR); or a vα -linker-vβ single chain, optionally fused to a self-cleaving peptide, e.g., a 2A-peptide. TCR expression with minimal modification of the wild-type amino acid sequence can also be optimized using the disclosed methods (e.g., szymczak, A.L. et al correction of multi-gene deficiency in vivo using a single 'self-clear' 2A peptide-based reduced vector. Nat. Biotechnol 22,589-594 (2004), yang, S.et al development of optimal bicistronic lentivi-ral vectors facilitates high-level TCR gene expression and robust tumor cell correction. Gene Ther 15,1411-1423 (2008), kuball, J.et al resolution matched pairing and ex-pression of TCR chains introduced into human T cells blood 109,2331-2338 (2007), cohen, C.J.et al enhanced Antitumor Activity of T Cells Engineered to Express T-Cell Receptors with a Second Disulfide bond. Cancer Res 67,3898-3903 (2007), K.B.J.et al cooling modification of T cell receptors allows enhanced functional expression in transgenic human T.135, cl 135-145).

The ratio of vector DNA to insert DNA is in the range of about 1:0.5 to about 1:10, e.g., 1:0.5, 1:1;1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In one embodiment, about 1. Mu.g of vector DNA and about 1. Mu.g of insert DNA are used in the reaction. In another embodiment, about 1 μg of vector DNA and about 2 μg of insert DNA are precipitated. In another embodiment, about 1 μg of vector DNA and about 3 μg of insert DNA are precipitated. In another embodiment, about 1 μg of vector DNA and about 4 μg of insert DNA are precipitated. In another embodiment, about 1 μg of vector DNA and about 5 μg of insert DNA are precipitated.

In one embodiment, the cell suspension comprises about 50 to about 400 μl of yeast cells, e.g., 50, 100, 150, 200, 250, 300, 350, 400 μl of yeast cells.

In one embodiment, the yeast cell suspension is from about 1 to about 10x 10 ⁹ Yeast cells/mL, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10x 10 ⁹ Individual yeast cells/mL.

In one embodiment, the field strength for electroporating the yeast cells is from about 0.5kV/cm to about 12.5kV/cm, e.g., 0.5, 1.0, about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5kV/cm.

In one embodiment, the yeast cells are electroporated with a capacitance of about 10 to about 50 μf, e.g., 10, 15, 20, 25, 30, 35, 40, 45, or 50.

In one embodiment, yeast cells are suspended in about 0.1 to about 10M sorbitol and 0.1 to 10mM CaCl ₂ Or MgCl ₂ For example 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0M sorbitol or, for example, 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0mM CaCl ₂ Or MgCl ₂ 。

In one embodiment, the yeast cells are incubated in about 0.01 to about 1.0M LiAc, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, about 0.7, 0.8, 0.9, or 1.0M LiAc, and 1 to 100mM TCEP, e.g., 1, 10, 20, 30, 40, 50, 60, about 70, 80, 90, or 100mM TCEP.

The invention provides methods of transforming yeast cells comprising electroporating a cell suspension comprising yeast and one or more nucleic acid constructs. Transformation of yeast cells can occur anywhere that a single clone into a population of yeast cells (i.e., a yeast library) can be used to screen for (a) peptides or proteins displayed on the surface of a yeast cell by ligating to a yeast surface protein, or by covalent or non-covalent interactions with a yeast cell surface protein or other component; (b) a peptide or protein expressed intracellularly; or (c) peptides or proteins secreted into the extracellular space (e.g., culture medium) or deposited on a solid surface. Such a yeast library may be conveniently adapted for use in a variety of applications to screen or characterize interactions between a peptide or protein and another protein, peptide, DNA, RNA or other chemical that may be introduced into a yeast cell or exogenously added. Specific examples are those found in yeast display, yeast two-hybrid, yeast three-hybrid, and the like.

The present invention provides a method for transforming a yeast cell comprising electroporating a cell suspension comprising yeast and one or more nucleic acid constructs comprising one or more regulatory sequences and one or more genes or gene fragments using one or more of electrical resistance, field strength, and pulse duration sufficient to transform the yeast cell.

In one embodiment, the field strength is from about 2.5kV/cm to about 12.5kV/cm. In certain embodiments, the field strength is 0.5kV/cm, 1.0kV/cm, 1.5kV/cm, 2.0kV/cm, or 2.5kV/cm. These values take into account that the electroporation cuvette has a gap of 0.2 cm. Higher field strengths are possible, but their operability is largely dependent on the development of devices capable of providing stronger pulses.

In one embodiment, the pulse duration is from about 3 milliseconds to about 10 milliseconds. In a particular embodiment, the pulse duration is about 4 milliseconds.

The treatment of cells by the electroporation method of the present invention is performed by applying an electric field to the yeast cell suspension between a pair of electrodes. The field strength must be adjusted fairly accurately so that electroporation of cells does not damage or minimizes damage to cells. The distance between the electrodes can then be measured and an appropriate voltage (e=electric field strength in V/cm; v=voltage in volts; d=distance in cm) can be applied to the electrodes according to the formula e=v/d.

Pulsers for performing the procedures described herein have been marketed for many years. One suitable signal generator is Gene Pulser II (Bio-Rad Laboratories, inc., hercules, calif.). Typical arrangements include a Gene Pulser II and a pulse controller+ module connected to a capacitance extender+.

Electroporation is used in the present invention to facilitate the introduction of DNA into yeast cells. Electroporation is a process of transiently permeabilizing a cell membrane using a pulsed electric field to allow large molecules (e.g., DNA molecules) to enter the cell. However, the actual mechanism of DNA transfer into cells is not yet known. For example, for the transformation of candida, the efficiency of electroporation is surprising when the cells are exposed to an experimentally decaying pulsed electric field having a field strength of about 10 to about 13kV/cm, a resistance value of about R5 (129 omu), a time constant of about 4.5 ms. Typically, depending on the electroporation device selected, the resistor and capacitor are off-the-shelf or selectable by the user. Regardless, the device is configured as per manufacturer's instructions to provide the appropriate field strength and attenuation parameters.

The invention also relates to methods for efficiently transforming yeast such that any one or more desired endogenous (i.e., naturally occurring within the yeast cell) or heterologous genes can be expressed at high levels. The methods of the invention also relate to methods of preparing libraries, e.g., libraries expressing TCRs, sctcrs, chimeras, or fragments thereof.

In one case, expression vectors carrying a gene associated with a sense can be transformed into yeast host cells by electroporation to produce single clones or libraries composed of a number of transformed cells expressing an intracellular protein (e.g., nuclear or cytoplasmic protein), a membrane protein (e.g., transmembrane or membrane-attached protein), or a secreted protein. One would be able to use transformed cells or libraries to purify proteins, study protein function, recognize protein-protein interactions, or recognize new protein conjugates or partners of interactions. Importantly, the ability to generate a very large library of yeasts displaying or expressing TCRs and TCR fragments. The library may be selected by the target antigen to find TCRs that bind to the selected antigen.

Since transformed yeasts have a tendency to lose artificial construction plasmids, it is advantageous to apply a positive selection pressure thereto using a medium. This selection pressure can be applied by omitting the metabolite in the culture medium when the strain is an auxotrophic mutant of an essential metabolite and when the vector plasmid used comprises a marker gene (e.g. the LEU2 gene) capable of restoring protoplasts of the strain. Other methods exist to achieve the same results and these methods may also be used to practice the present invention.

Depending on the nature of the structural gene concerned, the product or expression product may remain in the cytoplasm of the yeast host cell or be secreted or expressed. It has been found that not only the proteins remaining in the cells but also the secreted proteins are soluble. When the product or expression product remains in the yeast host cell, it may often be desirable to have an inducible transcription initiation region so that little or no expression or production of the desired product occurs until the transformant reaches high density. After the product or expression product has been expressed for a sufficient period of time, the cells may be separated by conventional methods (e.g., centrifugation, lysis) to isolate the relevant product. Depending on the nature and use of the product, the lysate may be subjected to various purification methods such as chromatography, electrophoresis, solvent extraction, crystallization, dialysis, ultrafiltration, etc. Chromatographic methods, including but not limited to gas chromatography, HPLC, column chromatography, ion exchange chromatography, and other chromatographic methods known to those of skill in the art. The purity may vary between about 50% to 90% or higher, preferably up to about 100%.

Alternatively, the expression product or related products may be secreted into the medium and continuously produced, wherein the medium is partially withdrawn, the desired product is extracted, for example by column or affinity chromatography, ultrafiltration, precipitation, etc., and the spent medium discarded or recycled by reduction of the essential components. The permeate containing the product from the ultrafiltration can be further concentrated, further crystallized or precipitated by evaporation, followed by alcohol and/or pH adjustment. Those skilled in the art are aware of many process options. When the product is secreted, a constitutive transcription initiation region is typically used, although non-constitutive regions may also be used.

Other preferred embodiments may be derived from the examples with reference to the figures as described herein, but are not limited thereto. For the purposes of the present invention, all references cited herein are incorporated by reference.

Drawings

FIG. 1 shows a comparison of the improved conversion efficiency of TCEP with DTT treatment under the same conditions.

Examples

The practice of the present invention employs, unless otherwise indicated, conventional techniques of cell electroporation and yeast cell biology, which are well known in the art.

I. Culture medium

YPD Medium

Yeast extract 10g

Bactopeptone 20g

Glucose 20g

By H ₂ O was adjusted to a volume of 1L (adding sterile glucose to the autoclaved solution)

2.SD-CAA(pH 4.5)：

Sodium citrate dihydrate 14.8g (50 mM final)

Citric acid monohydrate 4.2g (20 mM final)

At 800mL H ₂ O, autoclaving.

Glycine 5.0g

Yeast nitrogen base (free of amino acids) 6.7g

Glucose 20g

By H ₂ O adjusts the volume to 1L, and the solution is filtered aseptically

SD-CAA plate:

at 800mL H ₂ In O, the mixture was autoclaved and cooled to-55 ℃.

At 200ml H ₂ Aseptically filtering in O, adding to cooled autoclave solution

II preparation of electrically induced yeast cells

Mu.l of freshly thawed yeast stock from-80℃were streaked onto YPD agar plates and incubated for 2 days at 30 ℃. Individual colonies (whole colonies) were removed from the YPD agar plates and placed in 15ml YPD medium and shaken overnight at 30 ℃. The next morning, 10ml of culture was transferred to 100ml of fresh YPD medium and shaking continued for 7 hours at 30 ℃. Determination of OD ₆₀₀ 1L of cold YPD medium was added to adjust the OD ₆₀₀ 0.2. The shake flask was placed in a pre-chilled (4 ℃) shaker. The shaker was programmed to start heating (30 ℃) and shaking (250 rpm) 5 hours before the start of the working day. Incubation is carried outUp to OD ₆₀₀ Up to 1.5, typically starting 6 hours after the start of oscillation.

If not otherwise stated, the subsequent steps must be carried out on ice and with cooled solutions, tubes, cuvettes and centrifuges.

Cells were centrifuged at 2000g and 4℃for 3 min (50 ml in 10 Falcon tubes, 2 steps), pelleted and cooled with 25ml cold H ₂ O was washed twice and agglomerated for 2000g 3 minutes. Cells were treated with 25ml of 1M cold sorbitol, 1mM CaCl ₂ Washing; and centrifuged at 2000g for 3 minutes. Cells were resuspended in 25ml 100mM lithium acetate, 10mM TCEP. Aeration was performed using a 50ml Falcon tube with a filter cap; cells were incubated at 30℃for 30 minutes with shaking at 160rpm, placed on ice and centrifuged at 2000g and 4℃for 3 minutes. Cells were treated with 25ml cold 1M sorbitol/1 mM CaCl ₂ Washing; centrifugation at 2000g and 4℃for 3 min, washing with 25ml cold 1M sorbitol; centrifuge at 2,000g and 4℃for 3 min. Cells were suspended in cold 1M sorbitol conical tubes until the final volume of each electroporation reaction was 400. Mu.l. Electrotransport competent cells can be stored directly at-80 ℃. Prior to electroporation using the samples, the leaked salt was removed by centrifugation (2000 g,4 ℃,5 min) and washed twice with 1M cold sorbitol.

Electroporation of

Mu.l of cells were combined with 5-10. Mu.l of DNA (vector) in H ₂ Mix in O, hold on ice for 3 min and transfer to a pre-chilled 0.2cm electroporation cuvette. Cells were electroporated at 2.5kV using a BioRad micropulse generation electroporation system. The usual time constant is about 4ms, preferably 4ms. The electroporated cells were transferred to 10ml of a 1:1 sorbitol YPD medium mixture at 30℃for 1 hour without shaking. Cells were collected at room temperature for 2000g for 3 min and resuspended in 10ml SD-CAA at room temperature. Dilutions were performed to calculate diversity (1:10 ⁵ To 1:10 ⁷ ). Dilutions were incubated on SD-CAA plates containing kanamycin for 1 day at 30℃and 3 days at room temperature. The library was transferred to 100ml SD-CAA (preferably per electroporation of the library), and at 30 degrees C, 160rpm continued to shake for 24 hours. The expanded library can be used directly for induction or inStored at 4℃for two weeks. Can be stored frozen at-80deg.C in 30% glycerol for a long period.

By using optimal electroporation conditions, about 2X 10 can be routinely achieved ⁸ Yeast transformation efficiency of individual yeast transformants per μg of vector DNA (see FIG. 1). Since this conversion efficiency is achieved in a minimized cell volume (100 μl), it is well suited for use in automated and multi-well electroporation devices.

Claims

1. A method of preparing an electrotransport competent yeast cell comprising the steps of:

a) Growing yeast cells to OD ₆₀₀ A value of 1.0 to 2;

b) Washing the cells with cold water;

c) By a process comprising sorbitol and CaCl ₂ Is used for washing cells;

d) Incubating the cells with a solution comprising lithium acetate and tris- (2-carboxyethyl) phosphine (TCEP);

e) By a process comprising sorbitol and CaCl ₂ Is used for washing cells;

f) Resuspending the cells in a solution comprising sorbitol; and

g) Optionally, the cells are stored appropriately.

2. An electrotransport competent yeast cell obtained using the method according to claim 1.

3. A method of transfecting an electrotransfection competent yeast cell comprising the steps of:

a) Providing an electrotransformation competent yeast cell according to claim 2;

b) Washing the cells with a cold solution comprising sorbitol;

c) Mixing the cells with DNA to be transfected to form an electroporation premix;

d) Transferring the electroporation pre-mix into a suitable electroporation cuvette, and

e) Cells were electroporated at 2.5kV/cm to 12.5kV/cm for 2-5 ms.

4. The method of claim 3, wherein the DNA is linear or circular.

5. The method of claim 3 or 4, wherein the DNA comprises a library of DNA fragments encoding a library of proteins of interest.

6. The method of claim 5, wherein the library of DNA fragments is in the form of a yeast surface display library.

7. The method of claim 6, wherein the display library is a T Cell Receptor (TCR) library.

8. The method of claim 3, wherein the transfection efficiency is greater than 1x 10 ⁸ Each yeast transformant/. Mu.g vector DNA.

9. The method of claim 8, wherein transfection efficiency is greater than 2x 10 ⁸ Each yeast transformant/. Mu.g vector DNA.

10. A method for producing an improved library of target protein yeasts comprising the steps of:

a) Providing a transfected yeast cell prepared according to the method of any one of claims 3 to 7;

b) Diluting transfected cells in a sorbitol solution in a 1:1 mixture of growth medium;

c) Resuspending the cells in a suitable growth medium;

d) Optionally, dilution was performed to calculate diversity, and the dilutions were plated on SD-CAA plates containing kanamycin; and

e) Transferring the library from each electroporation to a suitable growth medium and expanding the library; and

f) Optionally, the expanded library is stored appropriately.

11. The method of claim 10, wherein the library is in the form of a yeast surface library.

12. The method of claim 10, wherein the library is a T cell receptor library.

13. The method of any one of claims 10-12, wherein the library has a diversity of greater than 10 ¹² 。